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Abstract:

According to the present invention, peptides comprising the amino acid
sequence of SEQ ID NO: 3, 4, 9, 23, 25, 30, 60, 63 or 68 were
demonstrated to have cytotoxic T lymphocyte (CTL) inducibility.
Therefore, the present invention provides a peptide having the amino acid
sequence selected from the group of SEQ ID NOs: 3, 4, 9, 23, 25, 30, 60,
63 and 68. The peptide can include one, two, or several amino acid
substitutions or addition so long as its CTL inducibility is retained.
Furthermore, the present invention provides pharmaceutical agents for
treating and/or prophylaxis of tumors, and/or prevention of postoperative
recurrence thereof, which comprises any of these peptides. The
pharmaceutical agents of this invention include vaccines.

4. The peptide of claim 3 having at least one substitution selected from
the group consisting of: (a) the second amino acid from the N-terminus is
selected from the group of phenylalanine, tyrosine, methionine and
tryptophan, and (b) the C-terminal amino acid is selected from the group
of phenylalanine, leucine, isoleucine, tryptophan and methionine.

5. A nonapeptide or decapeptide comprising of the amino acid sequence
selected from the group consisting of SEQ ID NO: 23, 25, 30, 60, 63 and
68.

7. The peptide of claim 6 having at least one substitution selected from
the group consisting of: (a) the second amino acid from the N-terminus is
leucine or methionine, and (b) the C-terminal amino acid is valine or
leucine.

8. A pharmaceutical agent for treating and/or prophylaxis of tumors,
and/or prevention of postoperative recurrence thereof, wherein the agent
comprises one or more of the following peptides: (a) an isolated
nonapeptide or decapeptide having cytotoxic T cell inducibility, wherein
said nonapeptide or decapeptide comprises an amino acid sequence selected
from the amino acid sequence of SEQ ID NO: 76; (b) a nonapeptide or
decapeptide comprising the amino acid sequence selected from the group
consisting of SEQ ID NOs: 3, 4 and 9; (c) a peptide having cytotoxic T
lymphocyte (CTL) inducibility, wherein the peptide comprises the amino
acid sequence selected from the group of SEQ ID NO: 3, 4 or 9, and SEQ ID
NO: 3, 4 or 9 wherein 1, 2, or several amino acids are substituted or
added; (d) a nonapeptide or decapeptide comprising of the amino acid
sequence selected from the group consisting of SEQ ID NO: 23, 25, 30, 60,
63 and 68; and/or (e) a peptide having CTL inducibility, wherein the
peptide comprises the amino acid sequence selected from the group of SEQ
ID NO: 23, 25, 30, 60, 63 or 68, and SEQ ID NO: 23, 25, 30, 60, 63 or 68
wherein 1, 2, or several amino acids are substituted or added, or a
polynucleotide encoding the peptide.

9. The pharmaceutical agent of claim 8, which is intended for the
administration to a subject whose HLA antigen is HLA-A24 or HLA-A02.

10. The pharmaceutical agent of claim 9, which is intended for treating
cancer.

11. The pharmaceutical agent of claim 10, which is a vaccine.

12. An exosome that presents on its surface a complex comprising any one
of the following peptides: (a) an isolated nonapeptide or decapeptide
having cytotoxic T cell inducibility, wherein said nonapeptide or
decapeptide comprises an amino acid sequence selected from the amino acid
sequence of SEQ ID NO: 76; (b) a nonapeptide or decapeptide comprising
the amino acid sequence selected from the group consisting of SEQ ID NOs:
3, 4 and 9; (c) a peptide having cytotoxic T lymphocyte (CTL)
inducibility, wherein the peptide comprises the amino acid sequence
selected from the group of SEQ ID NO: 3, 4 or 9, and SEQ ID NO: 3, 4 or 9
wherein 1, 2, or several amino acids are substituted or added; (d) a
nonapeptide or decapeptide comprising of the amino acid sequence selected
from the group consisting of SEQ ID NO: 23, 25, 30, 60, 63 and 68; and/or
(e) a peptide having CTL inducibility, wherein the peptide comprises the
amino acid sequence selected from the group of SEQ ID NO: 23, 25, 30, 60,
63 or 68, and SEQ ID NO: 23, 25, 30, 60, 63 or 68 wherein 1, 2, or
several amino acids are substituted or added, and an HLA antigen.

13. The exosome of claim 12, wherein the HLA antigen is HLA-A24.

14. The exosome of claim 12, wherein the HLA antigen is HLA-A2402.

15. The exosome of claim 12, wherein the HLA antigen is HLA-A02.

16. The exosome of claim 12, wherein the HLA antigen is HLA-A0201.

17. A method for inducing an antigen-presenting cell with high CTL
inducibility by using a peptide of one of the following peptides: (a) an
isolated nonapeptide or decapeptide having cytotoxic T cell inducibility,
wherein said nonapeptide or decapeptide comprises an amino acid sequence
selected from the amino acid sequence of SEQ ID NO: 76; (b) a nonapeptide
or decapeptide comprising the amino acid sequence selected from the group
consisting of SEQ ID NOs: 3, 4 and 9; (c) a peptide having cytotoxic T
lymphocyte (CTL) inducibility, wherein the peptide comprises the amino
acid sequence selected from the group of SEQ ID NO: 3, 4 or 9, and SEQ ID
NO: 3, 4 or 9 wherein 1, 2, or several amino acids are substituted or
added; (d) a nonapeptide or decapeptide comprising of the amino acid
sequence selected from the group consisting of SEQ ID NO: 23, 25, 30, 60,
63 and 68; or (e) a peptide having CTL inducibility, wherein the peptide
comprises the amino acid sequence selected from the group of SEQ ID NO:
23, 25, 30, 60, 63 or 68, and SEQ ID NO: 23, 25, 30, 60, 63 or 68 wherein
1, 2, or several amino acids are substituted or added, or a
polynucleotide encoding the peptide.

18. A method for inducing CTL by using any one of the following peptides:
(a) an isolated nonapeptide or decapeptide having cytotoxic T cell
inducibility, wherein said nonapeptide or decapeptide comprises an amino
acid sequence selected from the amino acid sequence of SEQ ID NO: 76; (b)
a nonapeptide or decapeptide comprising the amino acid sequence selected
from the group consisting of SEQ ID NOs: 3, 4 and 9; (c) a peptide having
cytotoxic T lymphocyte (CTL) inducibility, wherein the peptide comprises
the amino acid sequence selected from the group of SEQ ID NO: 3, 4 or 9,
and SEQ ID NO: 3, 4 or 9 wherein 1, 2, or several amino acids are
substituted or added; (d) a nonapeptide or decapeptide comprising of the
amino acid sequence selected from the group consisting of SEQ ID NO: 23,
25, 30, 60, 63 and 68; and/or (e) a peptide having CTL inducibility,
wherein the peptide comprises the amino acid sequence selected from the
group of SEQ ID NO: 23, 25, 30, 60, 63 or 68, and SEQ ID NO: 23, 25, 30,
60, 63 or 68 wherein 1, 2, or several amino acids are substituted or
added, or a polynucleotide encoding the peptide.

19. A method for inducing an antigen-presenting cell with high CTL
inducibility by using one of the following peptides: (a) an isolated
nonapeptide or decapeptide having cytotoxic T cell inducibility, wherein
said nonapeptide or decapeptide comprises an amino acid sequence selected
from the amino acid sequence of SEQ ID NO: 76; (b) a nonapeptide or
decapeptide comprising the amino acid sequence selected from the group
consisting of SEQ ID NOs: 3, 4 and 9; (c) a peptide having cytotoxic T
lymphocyte (CTL) inducibility, wherein the peptide comprises the amino
acid sequence selected from the group of SEQ ID NO: 3, 4 or 9, and SEQ ID
NO: 3, 4 or 9 wherein 1, 2, or several amino acids are substituted or
added; (d) a nonapeptide or decapeptide comprising of the amino acid
sequence selected from the group consisting of SEQ ID NO: 23, 25, 30, 60,
63 and 68; or (e) a peptide having CTL inducibility, wherein the peptide
comprises the amino acid sequence selected from the group of SEQ ID NO:
23, 25, 30, 60, 63 or 68, and SEQ ID NO: 23, 25, 30, 60, 63 or 68 wherein
1, 2, or several amino acids are substituted or added, or a
polynucleotide encoding the peptide, wherein said method comprises the
step of introducing a gene that comprises a polynucleotide encoding a
peptide listed above into an antigen-presenting cell.

20. An isolated cytotoxic T cell which targets any of the following
peptides: (a) an isolated nonapeptide or decapeptide having cytotoxic T
cell inducibility, wherein said nonapeptide or decapeptide comprises an
amino acid sequence selected from the amino acid sequence of SEQ ID NO:
76; (b) a nonapeptide or decapeptide comprising the amino acid sequence
selected from the group consisting of SEQ ID NOs: 3, 4 and 9; (c) a
peptide having cytotoxic T lymphocyte (CTL) inducibility, wherein the
peptide comprises the amino acid sequence selected from the group of SEQ
ID NO: 3, 4 or 9, and SEQ ID NO: 3, 4 or 9 wherein 1, 2, or several amino
acids are substituted or added; (d) a nonapeptide or decapeptide
comprising of the amino acid sequence selected from the group consisting
of SEQ ID NO: 23, 25, 30, 60, 63 and 68; and/or (e) a peptide having CTL
inducibility, wherein the peptide comprises the amino acid sequence
selected from the group of SEQ ID NO: 23, 25, 30, 60, 63 or 68, and SEQ
ID NO: 23, 25, 30, 60, 63 or 68 wherein 1, 2, or several amino acids are
substituted or added.

21. An isolated cytotoxic T cell that is induced by using any one of the
following peptides: (a) an isolated nonapeptide or decapeptide having
cytotoxic T cell inducibility, wherein said nonapeptide or decapeptide
comprises an amino acid sequence selected from the amino acid sequence of
SEQ ID NO: 76; (b) a nonapeptide or decapeptide comprising the amino acid
sequence selected from the group consisting of SEQ ID NOs: 3, 4 and 9;
(c) a peptide having cytotoxic T lymphocyte (CTL) inducibility, wherein
the peptide comprises the amino acid sequence selected from the group of
SEQ ID NO: 3, 4 or 9, and SEQ ID NO: 3, 4 or 9 wherein 1, 2, or several
amino acids are substituted or added; (d) a nonapeptide or decapeptide
comprising of the amino acid sequence selected from the group consisting
of SEQ ID NO: 23, 25, 30, 60, 63 and 68; and/or (e) a peptide having CTL
inducibility, wherein the peptide comprises the amino acid sequence
selected from the group of SEQ ID NO: 23, 25, 30, 60, 63 or 68, and SEQ
ID NO: 23, 25, 30, 60, 63 or 68 wherein 1, 2, or several amino acids are
substituted or added, or which is transduced with the nucleic acids
encoding the TCR subunits polypeptides binding with a peptide listed
above in the context of HLA-A24 or HLA-A2.

22. An isolated antigen-presenting cell that presents on its surface a
complex of an HLA antigen and one of the following peptides: (a) an
isolated nonapeptide or decapeptide having cytotoxic T cell inducibility,
wherein said nonapeptide or decapeptide comprises an amino acid sequence
selected from the amino acid sequence of SEQ ID NO: 76; (b) a nonapeptide
or decapeptide comprising the amino acid sequence selected from the group
consisting of SEQ ID NOs: 3, 4 and 9; (c) a peptide having cytotoxic T
lymphocyte CTL inducibility, wherein the peptide comprises the amino acid
sequence selected from the group of SEQ ID NO: 3, 4 or 9, and SEQ ID NO:
3, 4 or 9 wherein 1, 2, or several amino acids are substituted or added;
(d) a nonapeptide or decapeptide comprising of the amino acid sequence
selected from the group consisting of SEQ ID NO: 23, 25, 30, 60, 63 and
68; or (e) a peptide having CTL inducibility, wherein the peptide
comprises the amino acid sequence selected from the group of SEQ ID NO:
23, 25, 30, 60, 63 or 68, and SEQ ID NO: 23, 25, 30, 60, 63 or 68 wherein
1, 2, or several amino acids are substituted or added.

23. An antigen-presenting cell which is induced by using one of the
following peptides: (a) an isolated nonapeptide or decapeptide having
cytotoxic T cell inducibility, wherein said nonapeptide or decapeptide
comprises an amino acid sequence selected from the amino acid sequence of
SEQ ID NO: 76; (b) a nonapeptide or decapeptide comprising the amino acid
sequence selected from the group consisting of SEQ ID NOs: 3, 4 and 9;
(c) a peptide having cytotoxic T lymphocyte (CTL) inducibility, wherein
the peptide comprises the amino acid sequence selected from the group of
SEQ ID NO: 3, 4 or 9, and SEQ ID NO: 3, 4 or 9 wherein 1, 2, or several
amino acids are substituted or added; (d) a nonapeptide or decapeptide
comprising of the amino acid sequence selected from the group consisting
of SEQ ID NO: 23, 25, 30, 60, 63 and 68; (e) a peptide having CTL
inducibility, wherein the peptide comprises the amino acid sequence
selected from the group of SEQ ID NO: 23, 25, 30, 60, 63 or 68, and SEQ
ID NO: 23, 25, 30, 60, 63 or 68 wherein 1, 2, or several amino acids are
substituted or added, or introducing a gene that comprises a
polynucleotide encoding the peptide into an antigen presenting cell.

24. A method of inducing an immune response against tumor-associated
endothelia in a subject comprising administering to said subject a
vaccine comprising one of the following peptides: (a) an isolated
nonapeptide or decapeptide having cytotoxic T cell inducibility, wherein
said nonapeptide or decapeptide comprises an amino acid sequence selected
from the amino acid sequence of SEQ ID NO: 76; (b) a nonapeptide or
decapeptide comprising the amino acid sequence selected from the group
consisting of SEQ ID NOs: 3, 4 and 9; (c) a peptide having cytotoxic T
lymphocyte (CTL) inducibility, wherein the peptide comprises the amino
acid sequence selected from the group of SEQ ID NO: 3, 4 or 9, and SEQ ID
NO: 3, 4 or 9 wherein 1, 2, or several amino acids are substituted or
added; (d) a nonapeptide or decapeptide comprising of the amino acid
sequence selected from the group consisting of SEQ ID NO: 23, 25, 30, 60,
63 and 68; or (e) a peptide having CTL inducibility, wherein the peptide
comprises the amino acid sequence selected from the group of SEQ ID NO:
23, 25, 30, 60, 63 or 68, and SEQ ID NO: 23, 25, 30, 60, 63 or 68 wherein
1, 2, or several amino acids are substituted or added, an immunologically
active fragment thereof, or a polynucleotide encoding the peptide or the
fragment.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional
Application No. 60/911,194, filed on Apr. 11, 2007, the entire disclosure
of which is hereby incorporated herein by reference in its entirety for
all purposes.

TECHNICAL FIELD

Field of the Invention

[0002] The present invention relates to the field of biological science,
more specifically to the field of cancer therapy. In particular, the
present invention relates to TEM8 peptides that are extremely effective
as cancer vaccines, and drugs for treating and prevention of tumors.

BACKGROUND ART

[0003] It has been demonstrated that CD8 positive cytotoxic T lymphocytes
(CTLs) recognize epitope peptides derived from tumor-associated antigens
(TAAs) on major histocompatibility complex (MHC) class I molecule, and
then kill the tumor cells. Since the discovery of the melanoma antigen
(MAGE) family as the first example of TAAs, many other TAAs have been
discovered through immunological approaches (Boon T, Int J Cancer 1993
May 8, 54(2): 177-80; Boon T & van der Bruggen P, J Exp Med 1996 Mar. 1,
183(3): 725-9), and some of the TAAs are now in the process of clinical
development as immunotherapeutic targets.

[0006] Tumor angiogenesis is critically involved in the progression of
tumors. It has been already demonstrated that an effective vaccine
against tumor angiogenesis could be developed according to an endothelial
cell-based approach, targeting vascular endothelial growth factor
receptors (VEGFRs) 1 and 2, as HLA class I molecules are not
down-regulated on endothelial cells (Wada S et al., Cancer Res 2005 Jun.
1, 65(11): 4939-46; Ishizaki H et al., Clin Cancer Res 2006 Oct. 1,
12(19): 5841-9). Moreover, since these therapeutic targets are
tumor-independent, the depletion of vascular endothelial cells in the
tumor microenvironment could be effective against a variety of
malignancies. Furthermore, tumor endothelial cells are readily accessed
by lymphocytes in the bloodstream, and CTLs can directly damage
endothelial cells without the penetration of any other tissue type. In
addition, the lysis of even a small number of endothelial cells within
the tumor vasculature may result in the destruction of vessel integrity,
thus leading to the inhibition of numerous tumor cells (Folkman J, Nat
Med 1995 January, 1(1): 27-31). Therefore, tumor endothelial cells are a
good target for cancer immunotherapy. In order to suppress tumor
angiogenesis with a specific and efficient CTL response, an appropriate
target needs to be selected among molecules that are related to
angiogenesis.

[0007] Tumor endothelial markers (TEMs) including TEM8, have been found to
be specifically elevated in tumor-associated endothelium compared with
normal tissue (St Croix B et al., Science 2000 Aug. 18, 289(5482):
1197-202). The TEM8 transcript was expressed in lung and brain tumor and
liver metastasis. Therapy targeting TEM8 is applicable to a wide range of
tumor types. For example, WO 2005/048943 proposes the use of vaccines
comprising a vector encoding the extracellular domain of TEM8 with a
vaccine encoding tumor-associated antigen. However, this document fails
to provide any evidence that the introduction of the TEM8-expressing
vector resulted in the induction of CTLs against tumor-associated
endothelium, nor does it provide any information on the position of
epitopes within the TEM8 protein.

DISCLOSURE OF INVENTION

Summary of the Invention

[0008] It is important to improve the clinical efficacy for cancer
treatment targeting tumor microenvironment, especially for those
targeting tumor angiogenesis. The present invention focuses on tumor
blood vessels as the target for anti-tumor immunotherapy. In particular,
the invention targets the tumor endothelial marker 8 (TEM8) (GenBank
Accession No. NP--115584 (SEQ ID NO: 76) encoded by the gene of
GenBank Accession No. NM--032208 (SEQ ID NO: 75)), since TEM8 has
been thought to be expressed in vessels of a wide range of tumor types.
The present invention provides TEM8 gene products containing epitope
peptides that elicit CTLs specific to the corresponding molecules.
Peripheral blood mononuclear cells (PBMCs) obtained from a healthy donor
were stimulated using HLA-A*2402 or HLA-A*0201 binding candidate peptides
derived from TEM8. The present invention further provides established
CTLs that specifically recognize HLA-A24 or HLA-A02 positive target cells
pulsed with the respective candidate peptides, and HLA-A24 or HLA-A02
restricted epitope peptides that can induce potent and specific immune
responses against TEM8 expressed on tumor blood vessels. These results
demonstrate that TEM8 is strongly immunogenic and the epitopes thereof
are effective targets for tumor immunotherapy.

[0009] Accordingly, the present invention provides an isolated nonapeptide
or decapeptide having cytotoxic T cell inducibility, wherein said
nonapeptide or decapeptide comprises an amino acid sequence selected from
amino acid sequence of SEQ ID NO: 76. Specifically, the present invention
provides peptides comprising an amino acid sequence selected from the
group of SEQ ID NOs: 3, 4, 9, 23, 25, 30, 60, 63 and 68, and which have
CTL inducibility. The peptides of the invention encompass those wherein
one, two or more amino acids are substituted or added, so long as the
modified peptides retain the original CTL inducibility.

[0010] When administered to a subject, the present peptides are presented
on the surface of antigen-expressing cells and then induce CTLs targeting
the respective peptides. Therefore, according to an aspect of the present
invention, antigen-presenting cells and exosomes which present any of the
present peptides, as well as methods for inducing antigen-presenting
cells are also provided.

[0011] An anti-tumor immune response is induced by the administration of
the present TEM8 polypeptides or polynucleotide encoding the
polypeptides, as well as exosomes and antigen-presenting cells which
present the TEM8 polypeptides. Therefore, the present invention provides
pharmaceutical agents containing the polypeptides or polynucleotides
encoding them, as well as the exosomes and antigen-presenting cells as
their active ingredients. The pharmaceutical agents of the present
invention find use as vaccines.

[0012] Moreover, the present invention provides methods for treating
and/or prophylaxis of (i.e., preventing) cancers (tumors), and/or
prevention of postoperative recurrence thereof, as well as methods for
inducing CTLs, methods for inducing an immune response against
tumor-associated endothelia and also anti-tumor immunity, which methods
comprise the step of administering the TEM8 polypeptides, polynucleotides
encoding TEM8 polypeptides, exosomes or the antigen-presenting cells
presenting TEM8 polypeptides or the pharmaceutical agents of the
invention.

[0013] In addition, CTLs that target the present TEM polypeptides
strengthen the immune responses targeting tumor-associated endothelium.
Therefore, the present invention provides CTLs that target the present
TEM polypeptides. The CTLs of the invention also find use as vaccines
against cancer.

[0014] It is to be understood that both the foregoing summary of the
invention and the following detailed description are of exemplified
embodiments, and not restrictive of the invention or other alternate
embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 depicts photographs showing the results of IFN-gamma ELISPOT
assay on CTLs that were induced with peptides derived from TEM8. The CTLs
in the well numbers #5 and #6 stimulated with TEM8-A24-9-39 (SEQ ID NO:3)
(a), #6 with TEM8-A24-9-277 (SEQ ID NO:4) (b), #3 with TEM8-A24-10-277
(SEQ ID NO:9) (c), #3 with TEM8-A02-9-337 (SEQ ID NO: 23) (d), #6 with
TEM8-A02-9-338 (SEQ ID NO: 25) (e), #3 with TEM8-A02-9-278 (SEQ ID NO:
30) (f), #2 with TEM8-A02-10-338 (SEQ ID NO: 60) (g), #5 with
TEM8-A02-10-265 (SEQ ID NO: 63) (h) and #4 with TEM8-A02-10-333 (SEQ ID
NO: 68) (i) showed potent IFN-gamma production compared with the control
respectively. In contrast, as typical case of negative data (no
CTL-induction), it was not shown specific IFN-gamma production from the
CTL stimulated with TEM8-A02-9-207 (SEQ ID NO: 46) against peptide-pulsed
target cells (j). Most of the predicted peptides showed no CTL-induction,
therefore the positive data (CTL-induction) was focused upon in this
invention. The square on the well of these pictures indicated that the
cells from corresponding well were expanded to establish CTL lines. In
the figures, "+" indicated the IFN-gamma production against target cells
pulsed with the appropriate peptide, and "-" indicated the IFN-gamma
production against target cells not pulsed with any peptides.

[0017] FIG. 3 depicts line graphs showing the establishment of CTL clones
stimulated with TEM8-A24-9-277 (SEQ ID NO:4) (a) TEM8-A24-10-277 (SEQ ID
NO:9) (b), TEM8-A02-9-337 (SEQ ID NO: 23) (c), TEM8-A02-9-338 (SEQ ID NO:
25) (d) and TEM8-A02-10-265 (SEQ ID NO: 63) (e). CTL clones established
by stimulation with each peptide demonstrated potent IFN-gamma production
against target cells pulsed corresponding peptide. On the other hand, no
IFN-gamma production was shown against target cells not pulsed with any
peptides. In the figures, "+" indicated the IFN-gamma production against
target cells pulsed with the appropriate peptide, and "-" indicated the
IFN-gamma production against target cells not pulsed with any peptides.

[0018] FIG. 4 depicts line graphs showing specific CTL activity against
the target cells that endogenously express TEM8 and HLA-A*2402 or
HLA-A*0201. COS7 cells transfected with the full length TEM8 gene or with
corresponding HLA gene pulsing with inappropriate peptide derived from
TEM8 were prepared as control. (a) The CTL clone established with
TEM8-A24-9-277 (SEQ ID NO: 4) showed high specific CTL activity against
COS7 cells transfected with both TEM8 and HLA-A24 (black lozenge-mark).
On the other hand, no significant specific CTL activity was detected
against target cells expressing either HLA-A*2402 (open triangular mark)
or TEM8 (open circle). (b) The CTL clone established with TEM8-A02-10-265
(SEQ ID NO: 63) showed high specific CTL activity against COS7 cells
transfected with both TEM8 and HLA-A02 (black lozenge-mark). On the other
hand, no significant specific CTL activity was detected against target
cells expressing either HLA-A*0201 (open triangular mark) or TEM8 (open
circle).

[0019] FIG. 5 depicts in vivo immunogenicity and antitumor effects of
vaccination using TEM8-A24-9-277 peptide. (a) In vivo immunogenicity of
TEM8 epitope peptide was examined according to the protocol as described
in "Materials and Methods". BALB/c mice were injected with Incomplete
Freund's adjuvant (IFA)-conjugated TEM8-A24-9-277 (SEQ ID NO: 4) (M1-M5)
or IFA only (N1 and N2). In the figures, "+" indicated the IFN-gamma
production against target cells pulsed with peptide (black bar), and "-"
indicated the IFN-gamma production against target cells not pulsed with
any peptides (white bar). Splenocytes from the vaccinated mice produced
IFN-gamma against RLmale1 cells pulsed with TEM8-A24-9-277 (SEQ ID NO: 4)
without producing it against target cells not pulsed with any peptides.
SFC indicated spot forming cells. (b) Antitumor effects by vaccination
using TEM8 epitope peptide was tested as preventive setting.
IFA-conjugated with TEM8-A24-9-277 (SEQ ID NO: 4) (black triangular mark)
or no peptide (open lozenge-mark) was injected on day -7 and 0 into
BALB/c mice. 5×104 CT26, mouse colorectal cancer cell lines,
were injected s.c. into vaccinated mice on day 0. Tumor sizes are
represented as the mean of five mice. Significant difference of tumor
growth suppression was observed by the vaccination of epitope peptide (*;
P<0.05).

[0021] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues. The
terms apply to amino acid polymers in which one or more amino acid
residue is a modified residue, or a non-naturally occurring residue, such
as an artificial chemical mimetic of a corresponding naturally occurring
amino acid, as well as to naturally occurring amino acid polymers.

[0022] The term "amino acid" as used herein refers to naturally occurring
and synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that similarly function to the naturally occurring amino acids.
Naturally occurring amino acids are those encoded by the genetic code, as
well as those modified after translation in cells (e.g., hydroxyproline,
gamma-carboxyglutamate, and O-phosphoserine). The phrase "amino acid
analog" refers to compounds that have the same basic chemical structure
(an alpha carbon bound to a hydrogen, a carboxy group, an amino group,
and an R group) as a naturally occurring amino acid but have a modified R
group or modified backbones (e.g., homoserine, norleucine, methionine,
sulfoxide, methionine methyl sulfonium). The phrase "amino acid mimetic"
refers to chemical compounds that have different structures but similar
functions to general amino acids.

[0023] Amino acids may be referred to herein by their commonly known three
letter symbols or the one-letter symbols recommended by the IUPAC-IUB
Biochemical Nomenclature Commission.

[0024] The terms "gene", "polynucleotides", "nucleotides" and "nucleic
acids" are used interchangeably herein unless otherwise specifically
indicated and are similarly to the amino acids referred to by their
commonly accepted single-letter codes.

[0025] Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs.

II. Peptides

[0026] To demonstrate that peptides derived from TEM8 function as an
antigen recognized by cytotoxic T lymphocytes (CTLs), peptides derived
from TEM8 (GenBank Accession No. NP--115584 (SEQ ID NO: 76)) were
analyzed to determine whether they were antigen epitopes restricted by
HLA-A24 or HLA-A02 which are commonly encountered HLA alleles (Date Y et
al., Tissue Antigens 47: 93-101, 1996; Kondo A et al., J Immunol 155:
4307-12, 1995; Kubo R T et al., J Immunol 152: 3913-24, 1994). Candidates
of HLA-A24 and HLA-A02 binding peptides derived from TEM8 were identified
using the information on their binding affinities to HLA-A24 and HLA-A02.
After in vitro stimulation of T-cells by dendritic cells (DCs) loaded
with these peptides, CTLs were successfully established using each of the
following peptides.

TEM8-A24-9-39 (SEQ ID NO: 3),

TEM8-A24-9-277 (SEQ ID NO: 4),

TEM8-A24-10-277 (SEQ ID NO: 9),

TEM8-A02-9-337 (SEQ ID NO: 23),

TEM8-A02-9-338 (SEQ ID NO: 25),

TEM8-A02-9-278 (SEQ ID NO: 30),

TEM8-A02-10-338 (SEQ ID NO: 60),

TEM8-A02-10-265 (SEQ ID NO: 63) and

TEM8-A02-10-333 (SEQ ID NO: 68).

[0027] These established CTLs showed potent specific CTL activity against
target cells pulsed with respective peptides. These results demonstrate
that TEM8 is an antigen recognized by CTL and that the following peptides
are epitope peptides of TEM8 restricted by HLA-A24 or HLA-A02.

[0028] TEM8-A24-9-39 (SEQ ID NO: 3),

[0029] TEM8-A24-9-277 (SEQ ID NO: 4),

[0030] TEM8-A24-10-277 (SEQ ID NO: 9),

[0031] TEM8-A02-9-337 (SEQ ID NO: 23),

[0032] TEM8-A02-9-338 (SEQ ID NO: 25),

[0033] TEM8-A02-9-278 (SEQ ID NO: 30),

[0034] TEM8-A02-10-338 (SEQ ID NO: 60),

[0035] TEM8-A02-10-265 (SEQ ID NO: 63) and

[0036] TEM8-A02-10-333 (SEQ ID NO: 68).

[0037] Since the TEM8 gene is over expressed in most cancer patients, it
is a good target for immunotherapy with enhanced clinical efficacy. Thus,
the present invention provides nonapeptides (peptides consisting of nine
amino acid residues) and decapeptides (peptides consisting of ten amino
acid residues) of CTL-recognized epitopes from TEM8. In the present
invention, amino acid sequences of nonapeptides or decapeptides may be
selected from SEQ ID NO:76. Thus, present invention provides an isolated
peptide having cytotoxic T cell inducibility, wherein the peptide
comprises nine or ten contiguous amino acid sequence selected from the
amino acid sequence of SEQ ID NO:76. More specifically, in some
embodiments, the invention provides peptides consisting of the amino acid
sequence selected from the group of SEQ ID NOs: 3, 4, 9, 23, 25, 30, 60,
63 and 68.

[0038] Generally, software programs now available on the Internet, such as
those described in Parker K C et al., J Immunol 1994 Jan. 1, 152(1):
163-75, can be used to calculate the binding affinities between various
peptides and HLA antigens in silico. Binding affinity with HLA antigens
can be measured as described, for example, in Parker K C et al., J
Immunol 1994 Jan. 1, 152(1): 163-75; and Kuzushima K et al., Blood 2001,
98(6): 1872-81. The methods for determining binding affinity is
described, for example, in; Journal of Immunological Methods, 1995, 185:
181-190; Protein Science, 2000, 9: 1838-1846. Thus, the present invention
encompasses peptides of TEM8 which are determined to bind with HLA
antigens by such known programs.

[0039] Furthermore, these peptides of the present invention can be flanked
with additional amino acid residues so long as the peptide retains its
CTL inducibility. Such peptides with CTL inducibility are for example,
less than about 40 amino acids, often less than about 20 amino acids,
usually less than about 15 amino acids. The amino acid sequence flanking
the peptides consisting of the amino acid sequence selected from the
group of SEQ ID NOs: 3, 4, 9, 23, 25, 30, 60, 63 and 68 is not limited
and can be composed of any kind of amino acids so long as it does not
impair the CTL inducibility of the original peptide. Thus, the present
invention also provides peptides having CTL inducibility, which comprises
the amino acid sequence selected from the group of SEQ ID NOs: 3, 4, 9,
23, 25, 30, 60, 63 and 68.

[0040] Generally, it is known that modifications of one or more amino acid
in a protein do not influence the function of the protein, or in some
cases even enhance the desired function of the original protein. In fact,
modified peptides (i.e., peptides composed of an amino acid sequence
modified by substituting or adding one, two or several amino acid
residues to an original reference sequence) have been known to retain the
biological activity of the original peptide (Mark et al., Proc Natl Acad
Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10:
6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79:
6409-13). Thus, according to one embodiment of the invention, the peptide
having CTL inducibility of the present invention can be composed of the
amino acids comprising the amino acid sequence of SEQ ID NO: 3, 4, 9, 23,
25, 30, 60, 63 or 68, wherein one, two or even more amino acids are added
and/or substituted.

[0041] One of skill in the art will recognize that individual additions or
substitutions to an amino acid sequence which alters a single amino acid
or a small percentage of amino acids results in the conservation of the
properties of the original amino acid side-chain; it is thus referred to
as "conservative substitution" or "conservative modification", wherein
the alteration of a protein results in a protein with similar functions.
Conservative substitution tables providing functionally similar amino
acids are well known in the art. Examples of properties of amino acid
side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),
hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side
chains having the following functional groups or characteristics in
common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group
containing side-chain (S, T, Y); a sulfur atom containing side-chain (C,
M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a
base containing side-chain (R, K, H); and an aromatic containing
side-chain (H, F, Y, W). In addition, the following eight groups each
contain amino acids that are conservative substitutions for one another:

[0044] Such conservatively modified peptides are also considered to be
peptides of the present invention. However, the peptide of the present
invention is not restricted thereto and can include non-conservative
modifications, so long as the peptide retains the CTL inducibility.
Furthermore, the modified peptides do not exclude CTL inducible peptides
of polymorphic variants, interspecies homologues, and alleles of TEM8.

[0045] To retain the requisite CTL inducibility one can modify (add or
substitute) a small number (for example, 1, 2 or several) or a small
percentage of amino acids. Herein, the term "several" means 5 or fewer
amino acids, for example, 3 or fewer. The percentage of amino acids to be
modified can be 20% or less, for example, 15% of less, for example 10% or
1 to 5%.

[0047] When used in immunotherapy, the present peptides are presented on
the surface of a cell or exosome as a complex with an HLA antigen.
Therefore, one can select peptides that possess high binding affinity to
the HLA antigen in addition to their CTL inducibility. Moreover, the
peptides can be modified by substitution, addition and such of the amino
acid residues to achieve a higher binding affinity. In addition to
peptides that are naturally displayed, since the regularity of the
sequences of peptides displayed by binding to HLA antigens is already
known (J Immunol 1994, 152: 3913; Immunogenetics 1995, 41: 178; J Immunol
1994, 155: 4307), modifications based on such regularity can be
introduced into the immunogenic peptides of the invention. For example,
peptides showing high HLA-A24 binding affinity have their second amino
acid from the N-terminus substituted with phenylalanine, tyrosine,
methionine, or tryptophan, and peptides whose amino acid at the
C-terminus is substituted with phenylalanine, leucine, isoleucine,
tryptophan, or methionine can also be favorably used. Thus, peptides
having the amino acid sequences of SEQ ID NOs: 3, 4 or 9 wherein the
second amino acid from the N-terminus is substituted with phenylalanine,
tyrosine, methionine, or tryptophan, and peptides, and/or wherein the
C-terminus is substituted with phenylalanine, leucine, isoleucine,
tryptophan, or methionine are encompassed by the present invention.

[0048] On the other hand, peptides which second amino acid from the
N-terminus is substituted with leucine or methionine, and in which the
C-terminal amino acid is substituted with valine or leucine can be used
as peptides with high HLA-02 binding affinity. Thus, peptides having any
of the amino acid sequences SEQ ID NO: 23, 25, 30, 60, 63 and 68 wherein
the second amino acid from the N-terminus is substituted with leucine or
methionine, and/or wherein the C-terminus is substituted with valine or
leucine are encompassed by the present invention. Substitutions can be
introduced not only at the terminal amino acids but also at the position
of potential TCR recognition of peptides. Several studies have
demonstrated that amino acid substitutions in a peptide can be equal to
or better than the original, for example CAP1, p53.sub.(264-272),
Her-2/neu.sub.(369-377) or gp100.sub.(209-217) (Zaremba et al. Cancer
Res. 57, 4570-4577, 1997, T. K. Hoffmann et al. J. Immunol. (2002) Feb.
1; 168(3):1338-47., S. O. Dionne et al. Cancer Immunol immunother. (2003)
52: 199-206 and S. O. Dionne et al. Cancer Immunology, Immunotherapy
(2004) 53, 307-314).

[0049] Furthermore, one to two amino acids can also be added to the N
and/or C-terminus of the present peptides. Such modified peptides with
high HLA antigen binding affinity and retained CTL inducibility are also
included in the present invention.

[0050] However, when the peptide sequence is identical to a portion of the
amino acid sequence of an endogenous or exogenous protein having a
different function, side effects such as autoimmune disorders or allergic
symptoms against specific substances may be induced. Therefore, one can
perform homology searches using available databases to avoid situations
in which the sequence of the peptide matches the amino acid sequence of
another protein. When it becomes clear from the homology searches that
there exists not even a peptide with 1 or 2 amino acids difference to the
objective peptide, the objective peptide can be modified in order to
increase its binding affinity with HLA antigens, and/or increase its CTL
inducibility without any danger of such side effects.

[0051] Although peptides having high binding affinity to the HLA antigens
as described above are expected to be highly effective, the candidate
peptides, which are selected according to the presence of high binding
affinity as an indicator, are further examined for the presence of CTL
inducibility. Herein, the phrase "CTL inducibility" indicates the ability
of the peptide to induce CTLs when presented on antigen-presenting cells.
Further, "CTL inducibility" includes the ability of the peptide to induce
CTL activation, CTL proliferation, promote CTL lysis of target cells, and
to increase CTL IFN-gamma production.

[0052] Confirmation of CTL inducibility is accomplished by inducing
antigen-presenting cells carrying human MHC antigens (for example,
B-lymphocytes, macrophages, and dendritic cells (DCs)), or more
specifically DCs derived from human peripheral blood mononuclear
leukocytes, and after stimulation with the peptides, mixing with
CD8-positive cells, and then measuring the IFN-gamma produced and
released by CTL against the target cells. As the reaction system,
transgenic animals that have been produced to express a human HLA antigen
(for example, those described in BenMohamed L, Krishnan R, Longmate J,
Auge C, Low L, Primus J, Diamond D J, Hum Immunol 2000 August, 61(8):
764-79, Related Articles, Books, Linkout Induction of CTL response by a
minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on
HLA class II restricted T(H) response) can be used. For example, the
target cells can be radiolabeled with 51Cr and such, and cytotoxic
activity can be calculated from radioactivity released from the target
cells. Alternatively, it can be examined by measuring IFN-gamma produced
and released by CTL in the presence of antigen-presenting cells (APCs)
that carry immobilized peptides, and visualizing the inhibition zone on
the media using anti-IFN-gamma monoclonal antibodies.

[0053] As a result of examining the CTL inducibility of the peptides as
described above, those having high binding affinity to an HLA antigen did
not necessarily have high inducibility. Furthermore, nonapeptides or
decapeptides selected from peptides comprising the amino acid sequences
indicated by SEQ ID NOs: 3, 4, 9, 23, 25, 30, 60, 63 and 68, showed
particularly high CTL inducibility as well as high binding affinity to an
HLA antigen. Thus, these peptides are exemplified embodiments of the
present invention.

[0054] In addition to modification of the present peptides, discussed
above, the peptides of the present invention can be further linked to
other substances, so long as they retain the CTL inducibility.
Exemplified substances include: peptides, lipids, sugar and sugar chains,
acetyl groups, natural and synthetic polymers, etc. The peptides can
contain modifications such as glycosylation, side chain oxidation, or
phosphorylation; so long as the modifications do not destroy the
biological activity of the peptides as described herein. These kinds of
modifications can be performed to confer additional functions (e.g.,
targeting function, and delivery function) or to stabilize the
polypeptide.

[0055] For example, to increase the in vivo stability of a polypeptide, it
is known in the art to introduce D-amino acids, amino acid mimetics or
unnatural amino acids; this concept can also be adopted for the present
polypeptides. The stability of a polypeptide can be assayed in a number
of ways. For instance, peptidases and various biological media, such as
human plasma and serum, can be used to test stability (see, e.g., Verhoef
et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302).

[0056] Herein, the peptides of the present invention can also be described
as "TEM8 peptide(s)" or "TEM8 polypeptide(s)".

III. Preparation of TEM8 Peptides

[0057] The peptides of the invention can be prepared using well known
techniques. For example, the peptides can be prepared synthetically, by
recombinant DNA technology or chemical synthesis. Peptide of the
invention can be synthesized individually or as longer polypeptides
comprising two or more peptides. The peptides can be isolated i.e.,
purified or isolated substantially free of other naturally occurring host
cell proteins and fragments thereof, or any other chemical substances.

[0066] Alternatively, the present peptides can be obtained adopting any
known genetic engineering methods for producing peptides (e.g., Morrison
J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in
Enzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first, a
suitable vector harboring a polynucleotide encoding the objective peptide
in an expressible form (e.g., downstream of a regulatory sequence
corresponding to a promoter sequence) is prepared and transformed into a
suitable host cell. The host cell is then cultured to produce the peptide
of interest. The peptide can also be produced in vitro adopting an in
vitro translation system.

IV. Polynucleotides

[0067] The present invention provides a polynucleotide which encodes any
of the aforementioned peptides of the present invention. These include
polynucleotides derived from the natural occurring TEM8 gene (GenBank
Accession No. NM--032208 (SEQ ID NO: 75)) and those having a
conservatively modified nucleotide sequence thereof. Herein, the phrase
"conservatively modified nucleotide sequence" refers to sequences which
encode identical or essentially identical amino acid sequences. Because
of the degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at
every position where an alanine is specified by a codon, the codon can be
altered to any of the corresponding codons described without altering the
encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of conservatively modified variations.
Every nucleic acid sequence herein which encodes a peptide also describes
every possible silent variation of the nucleic acid. One of skill will
recognize that each codon in a nucleic acid (except AUG, which is
ordinarily the only codon for methionine, and TGG, which is ordinarily
the only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic acid
that encodes a peptide is implicitly described in each disclosed
sequence.

[0068] The polynucleotide of the present invention can be composed of DNA,
RNA, and derivatives thereof. A DNA is suitably composed of bases such as
A, T, C, and G, and T is replaced by U in an RNA.

[0069] The polynucleotide of the present invention can encode multiple
peptides of the present invention with or without intervening amino acid
sequences in between. For example, the intervening amino acid sequence
can provide a cleavage site (e.g., enzyme recognition sequence) of the
polynucleotide or the translated peptides. Furthermore, the
polynucleotide can include any additional sequences to the coding
sequence encoding the peptide of the present invention. For example, the
polynucleotide can be a recombinant polynucleotide that includes
regulatory sequences required for the expression of the peptide or can be
an expression vector (plasmid) with marker genes and such. In general,
such recombinant polynucleotides can be prepared by the manipulation of
polynucleotides through conventional recombinant techniques using, for
example, polymerases and endonucleases.

[0070] Both recombinant and chemical synthesis techniques can be used to
produce the polynucleotides of the present invention. For example, a
polynucleotide can be produced by insertion into an appropriate vector,
which can be expressed when transfected into a competent cell.
Alternatively, a polynucleotide can be amplified using PCR techniques or
expression in suitable hosts (see, e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York,
1989). Alternatively, a polynucleotide can be synthesized using the solid
phase techniques, as described in Beaucage S L & Iyer R P, Tetrahedron
1992, 48: 2223-311; Matthes et al., EMBO J. 1984, 3: 801-5.

V. Exosomes

[0071] The present invention further provides intracellular vesicles
called exosomes, which present complexes formed between the peptides of
this invention and HLA antigens on their surface. Exosomes can be
prepared, for example by using the methods detailed in Japanese Patent
Application Kohyo Publications Nos. Hei 11-510507 and WO99/03499, and can
be prepared using APCs obtained from patients who are subject to
treatment and/or prevention. The exosomes of this invention can be
inoculated as vaccines, similarly to the peptides of this invention.

[0072] The type of HLA antigens comprised in the complexes must match that
of the subject requiring treatment and/or prevention. For example, for
Japanese, HLA-A24, particularly HLA-A2402 is often appropriate. The use
of A-24 or A-02 type that are highly expressed among the Japanese and
Caucasian is favorable for obtaining effective results, and subtypes such
as A-2402 and A-0201 find use. Typically, in the clinic, the type of HLA
antigen of the patient requiring treatment is investigated in advance,
which enables appropriate selection of peptides having high levels of
binding affinity to this antigen, or having CTL inducibility by antigen
presentation. Furthermore, in order to obtain peptides showing high
binding affinity and CTL inducibility, substitution or addition of 1, 2,
or several amino acids can be performed based on the amino acid sequence
of the naturally occurring TEM8 partial peptide.

[0073] In case of using A-24 type HLA antigen for the exosome of the
present invention, the peptides comprising the sequence of SEQ ID NO: 3,
4, or 9 find use, whereas in case of using A-02 type HLA antigen, those
comprising the sequence of SEQ ID NO: 23, 25, 30, 60, 63 or 68 find use.

VI. Antigen-Presenting Cells (APCs)

[0074] The present invention also provides APCs that present complexes
formed between HLA antigens and the peptides of this invention on its
surface. The APCs that are obtained by contacting the peptides of this
invention, or introducing the nucleotides encoding the peptides of this
invention in an expressible form can be derived from patients who are
subject to treatment and/or prevention, and can be administered as
vaccines by themselves or in combination with other drugs including the
peptides of this invention, exosomes, or cytotoxic T cells.
Alternatively, the present invention also provides APCs presenting the
peptide of the present invention with HLA antigens, wherein said APCs is
induced by;

(a) contacting the peptides of the present invention with APCs, or (b)
introducing a polynucleotide encoding the peptides into APCs to produce
the APCs.

[0075] The APCs are not limited to a particular kind of cells and include
DCs, Langerhans cells, macrophages, B cells, and activated T cells, which
are known to present proteinaceous antigens on their cell surface so as
to be recognized by lymphocytes. Since DC is a representative APC having
the strongest CTL inducing action among APCs, DCs find use as the APCs of
the present invention.

[0076] For example, an APC can be obtained by inducing DCs from peripheral
blood monocytes and then contacting (stimulating) them with the peptides
of this invention in vitro, ex vivo or in vivo. When the peptides of this
invention are administered to the subjects, APCs that present the
peptides of this invention are induced in the body of the subject.
"Inducing APC" includes contacting (stimulating) a cell with the peptides
of this invention, or nucleotides encoding the peptides of this invention
to present complexes formed between HLA antigens and the peptides of this
invention on cell's surface. Alternatively, after introducing the
peptides of this invention to the APCs to allow the APCs to present the
peptides, the APCs can be administered to the subject as a vaccine. For
example, the ex vivo administration can comprise steps of:

[0077] a: collecting APCs from a first subject,

[0078] b: contacting with the APCs of step a, with the peptide and

[0079] c: administering the peptide-loaded APCs to a second subject.

[0080] The first subject and the second subject can be the same
individual, or can be different individuals. Alternatively, according to
the present invention, use of the peptides of this invention for
manufacturing a pharmaceutical composition inducing antigen-presenting
cells is provided. Further, the present invention also provides the
peptides of the present invention for inducing antigen-presenting cells.
The APCs obtained by step b can be administered to the subject as a
vaccine.

[0081] According to an aspect of the present invention, the APCs have a
high level of CTL inducibility. In the term of "high level of CTL
inducibility", the high level is relative to the level of that by APC
contacting with no peptide or peptides which can not induce the CTL. Such
APCs having a high level of CTL inducibility can be prepared by a method
which comprises the step of transferring genes comprising polynucleotides
that encode the peptides of this invention to APCs in vitro. The
introduced genes can be in the form of DNAs or RNAs. Examples of methods
for introduction include, without particular limitations, various methods
conventionally performed in this field, such as lipofection,
electroporation, and calcium phosphate method can be used. More
specifically, it can be performed as described in Cancer Res 1996, 56:
5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72;
Published Japanese Translation of International Publication No.
2000-509281. By transferring the gene into APCs, the gene undergoes
transcription, translation, and such in the cell, and then the obtained
protein is processed by MHC Class I or Class II, and proceeds through a
presentation pathway to present partial peptides.

VII. Cytotoxic T Cells

[0082] A cytotoxic T cell induced against any of the peptides of the
present invention strengthens the immune response targeting
tumor-associated endothelia in vivo and thus can be used as vaccines
similar to the peptides. Thus, the present invention provides isolated
cytotoxic T cells that are specifically induced or activated by any of
the present peptides. Preferably, the present invention provides an
isolated cytotoxic T cell;

[0083] (a) that is induced by the step of contacting CD8+ T cells
with APCs presenting the peptides of the present invention with HLA
antigens, or

[0084] (b) which is transduced with the nucleic acids encoding the TCR
subunits polypeptides binding with a peptide the present invention in the
context of HLA-A24 or HLA-A2.

[0085] Such cytotoxic T cells can be obtained by (1) administering to a
subject or (2) contacting (stimulating) subject-derived APCs, and
CD8-positive cells, or peripheral blood mononuclear leukocytes in vitro
with the peptides of the present invention.

[0086] The cytotoxic T cells, which have been induced by stimulation from
APCs that present the peptides of this invention, can be derived from
patients who are subject to treatment and/or prevention, and can be
administered by themselves or in combination with other drugs including
the peptides of this invention or exosomes for the purpose of regulating
effects. The obtained cytotoxic T cells act specifically against target
cells presenting the peptides of this invention, or for example, the same
peptides used for induction. The target cells can be cells that
endogenously express TEM8, or cells that are transfected with the TEM8
gene; and cells that present a peptide of this invention on the cell
surface due to stimulation by the peptide can also serve as targets of
activated CTL attack.

VIII. T Cell Receptor (TCR)

[0087] The present invention also provides a composition comprising
nucleic acids encoding polypeptides that are capable of forming a subunit
of a T cell receptor (TCR), and methods of using the same. The TCR
subunits have the ability to form TCRs that confer specificity to T cells
against tumor cells presenting TEM8. By using the known methods in the
art, the nucleic acids of alpha- and beta-chains as the TCR subunits of
the CTL induced with one or more peptides of this invention can be
identified (WO2007/032255 and Morgan et al., J Immunol, 171, 3288
(2003)). The derivative TCRs can bind target cells displaying the TEM8
peptide with high avidity, and optionally mediate efficient killing of
target cells presenting the TEM8 peptide in vivo and in vitro.

[0088] The nucleic acids encoding the TCR subunits can be incorporated
into suitable vectors e.g. retroviral vectors. These vectors are well
known in the art. The nucleic acids or the vectors comprising them
usefully can be transferred into a T cell, for example, a T cell from a
patient. Advantageously, the invention provides an off-the-shelf
composition allowing rapid modification of a patient's own T cells (or
those of another mammal) to rapidly and easily produce modified T cells
having excellent cancer cell killing properties.

[0089] Also, the present invention provides CTLs which are prepared by
transduction with the nucleic acids encoding the TCR subunits
polypeptides that bind to the TEM8 peptide e.g. SEQ ID NOs: 3, 4, 9, 23,
25, 30, 60, 63 and 68 in the context of HLA-A24 or HLA-A2. The transduced
CTLs are capable of homing to cancer cells in vivo, and can be expanded
by well known culturing methods in vitro (e.g., Kawakami et al., J
Immunol., 142, 3452-3461 (1989)). The T cells of the invention can be
used to form an immunogenic composition useful in treating or the
prevention of cancer in a patient in need of therapy or protection
(WO2006/031221).

[0090] Prevention and prophylaxis include any activity which reduces the
burden of mortality or morbidity from disease. Prevention and prophylaxis
can occur "at primary, secondary and tertiary prevention levels." While
primary prevention and prophylaxis avoid the development of a disease,
secondary and tertiary levels of prevention and prophylaxis encompass
activities aimed at the prevention and prophylaxis of the progression of
a disease and the emergence of symptoms as well as reducing the negative
impact of an already established disease by restoring function and
reducing disease-related complications. Alternatively, prevention and
prophylaxis include a wide range of prophylactic therapies aimed at
alleviating the severity of the particular disorder, e.g. reducing the
proliferation and metastasis of tumors, reducing angiogenesis.

[0091] Treating and/or for the prophylaxis of cancer or, and/or the
prevention of postoperative recurrence thereof includes any of the
following steps, such as surgical removal of cancer cells, inhibition of
the growth of cancerous cells, involution or regression of a tumor,
induction of remission and suppression of occurrence of cancer, tumor
regression, and reduction or inhibition of metastasis. Effectively
treating and/or the prophylaxis of cancer decreases mortality and
improves the prognosis of individuals having cancer, decreases the levels
of tumor markers in the blood, and alleviates detectable symptoms
accompanying cancer. For example, reduction or improvement of symptoms
constitutes effectively treating and/or the prophylaxis include 10%, 20%,
30% or more reduction, or stable disease.

IX. Pharmaceutical Agents

[0092] Since TEM8 expression is specifically elevated in tumor-associated
endothelium compared with normal tissue (St Croix B et al., Science 2000
Aug. 18, 289(5482): 1197-202), the peptides of this invention or
polynucleotides encoding the peptides can be used for treating and/or for
the prophylaxis of cancer, and/or prevention of postoperative recurrence
thereof. Thus, the present invention provides a pharmaceutical agent for
treating and/or for the prophylaxis of cancer, and/or prevention of
postoperative recurrence thereof, which comprises one or more of the
peptides of this invention, or polynucleotides encoding the peptides as
an active ingredient. Alternatively, the present peptides can be
expressed on the surface of any of the foregoing exosomes or cells, such
as APCs for the use as pharmaceutical agents. In addition, the
aforementioned cytotoxic T cells which target any of the peptides of the
invention can also be used as the active ingredient of the present
pharmaceutical agents.

[0093] The present pharmaceutical agents find use as a vaccine. In the
present invention, the phrase "vaccine" (also referred to as an
immunogenic composition) refers to a substance that has the function to
induce anti-tumor immunity upon inoculation into animals.

[0094] The pharmaceutical agents of the present invention can be used to
treat and/or prevent cancers, and/or prevention of postoperative
recurrence thereof in subjects or patients including human and any other
mammal including, but not limited to, mouse, rat, guinea-pig, rabbit,
cat, dog, sheep, goat, pig, cattle, horse, monkey, baboon, and
chimpanzee, particularly a commercially important animal or a
domesticated animal.

[0095] According to the present invention, polypeptides comprising the
amino acid sequence of SEQ ID NO: 3, 4 or 9 have been found to be HLA-A24
restricted epitope peptides that can induce potent and specific immune
response against tumor-associated endothelium. Therefore, the present
pharmaceutical agents which comprise any of these polypeptides with the
amino acid sequences of SEQ ID NOs: 3, 4 and 9 are particularly suited
for the administration to subjects whose HLA antigen is HLA-A24. On the
other hand, the polypeptides comprising the amino acid sequence of SEQ ID
NO: 23, 25, 30, 60, 63 or 68 have been found to be HLA-A02 restricted
epitope peptides that can induce potent and specific immune response
against tumor-associated endothelium. Therefore, the pharmaceutical
agents which comprise any of these polypeptides that comprise any of
these polypeptides with the amino acid sequences of SEQ ID NOs: 23, 25,
30, 60, 63 and 68 are particularly suited for the administration to
subjects whose HLA antigen is HLA-A02. The same applies to pharmaceutical
agents which comprise polynucleotides encoding any of these polypeptides.

[0097] The present pharmaceutical agents can contain in addition to the
aforementioned active ingredients, other peptides which have the ability
to induce CTLs against cancerous cells, other polynucleotides encoding
the other peptides, other cells that present the other peptides, or such.
Herein, the other peptides that have the ability to induce CTLs against
cancerous cells are exemplified by cancer specific antigens (e.g.,
identified TAAs), but are not limited thereto.

[0098] If needed, the pharmaceutical agents of the present invention can
optionally include other therapeutic substances as an active ingredient,
so long as the substance does not inhibit the antitumoral effect on
tumor-associated endothelium of the active ingredient, e.g., any of the
present peptides. For example, formulations can include anti-inflammatory
agents, pain killers, chemotherapeutics, and the like. In addition to
including other therapeutic substances in the medicament itself, the
medicaments of the present invention can also be administered
sequentially or concurrently with the one or more other pharmacologic
agents. The amounts of medicament and pharmacologic agent depend, for
example, on what type of pharmacologic agent(s) is/are used, the disease
being treated, and the scheduling and routes of administration.

[0099] It should be understood that in addition to the ingredients
particularly mentioned herein, the pharmaceutical agents of this
invention can include other agents conventional in the art having regard
to the type of formulation in question.

[0100] In one embodiment of the present invention, the present
pharmaceutical agents can be included in articles of manufacture and kits
containing materials useful for treating the pathological conditions of
the disease to be treated, e.g, cancer. The article of manufacture can
include a container of any of the present pharmaceutical agents with a
label. Suitable containers include bottles, vials, and test tubes. The
containers can be formed from a variety of materials, such as glass or
plastic. The label on the container should indicate the agent is used for
treating or prevention of one or more conditions of the disease. The
label can also indicate directions for administration and so on.

[0101] In addition to the container described above, a kit including a
pharmaceutical agent of the present invention can optionally further
include a second container housing a pharmaceutically-acceptable diluent.
It can further include other materials desirable from a commercial and
user standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.

[0102] The pharmaceutical compositions can, if desired, be presented in a
pack or dispenser device which can contain one or more unit dosage forms
containing the active ingredient. The pack can, for example, include
metal or plastic foil, such as a blister pack. The pack or dispenser
device can be accompanied by instructions for administration.

[0104] The peptides of this invention can be administered directly as a
pharmaceutical agent, or if necessary, that has been formulated by
conventional formulation methods. In the latter case, in addition to the
peptides of this invention, carriers, excipients, and such that are
ordinarily used for drugs can be included as appropriate without
particular limitations. Examples of such carriers are sterilized water,
physiological saline, phosphate buffer, culture fluid and such.
Furthermore, the pharmaceutical agents can contain as necessary,
stabilizers, suspensions, preservatives, surfactants and such. The
pharmaceutical agents of this invention can be used for anticancer
purposes.

[0105] The peptides of this invention can be prepared in a combination,
which comprises two or more of peptides of the invention, to induce CTL
in vivo. The peptides can be in a cocktail or can be conjugated to each
other using standard techniques. For example, the peptides can be
chemically linked or expressed as a single fusion polypeptide sequence.
The peptides in the combination can be the same or different. By
administering the peptides of this invention, the peptides are presented
at a high density by the HLA antigens on APCs, then CTLs that
specifically react toward the complex formed between the displayed
peptide and the HLA antigen are induced. Alternatively, APCs that present
any of the peptides of this invention on their cell surface are obtained
by removing APCs (e.g., DCs) from the subjects, which are stimulated by
the peptides of this invention, CTL is induced in the subjects by
readministering these APCs (e.g., DCs) to the subjects, and as a result,
aggressiveness towards the tumor-associated endothelium can be increased.

[0106] The pharmaceutical agents for treating and/or prevention of cancer,
which comprise a peptide of this invention as the active ingredient, can
comprise an adjuvant so that cellular immunity will be established
effectively, or they can be administered with other active ingredients,
and they can be administered by formulation into granules. An adjuvant
refers to a compound that enhances the immune response against the
protein when administered together (or successively) with the protein
having immunological activity. An adjuvant that can be applied includes
those described in the literature (Clin Microbiol Rev 1994, 7: 277-89).
Exemplary adjuvants include aluminum phosphate, aluminum hydroxide, alum,
cholera toxin, salmonella toxin, and such, but are not limited thereto.

[0107] Furthermore, liposome formulations, granular formulations in which
the peptide is bound to few-micrometers diameter beads, and formulations
in which a lipid is bound to the peptide may be conveniently used.

[0108] In some embodiments, the pharmaceutical agents of the invention
comprise a component which primes CTL. Lipids have been identified as
agents capable of priming CTL in vivo against viral antigens. For
example, palmitic acid residues can be attached to the epsilon- and
alpha-amino groups of a lysine residue and then linked to a peptide of
the invention. The lipidated peptide can then be administered either
directly in a micelle or particle, incorporated into a liposome, or
emulsified in an adjuvant. As another example of lipid priming of CTL
responses, E. coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to prime
CTL when covalently attached to an appropriate peptide (see, e.g., Deres
et al., Nature 1989, 342: 561-4).

[0109] The method of administration can be oral, intradermal,
subcutaneous, intravenous injection, or such, and systemic administration
or local administration to the vicinity of the targeted sites. The
administration can be performed by single administration or boosted by
multiple administrations. The dose of the peptides of this invention can
be adjusted appropriately according to the disease to be treated, age of
the patient, weight, method of administration, and such, and is
ordinarily 0.001 mg to 1000 mg, for example, 0.001 mg to 1000 mg, for
example, 0.1 mg to 10 mg, and can be administered once in a few days to
few months. One skilled in the art can appropriately select a suitable
dose.

[0111] The pharmaceutical agents of the invention can also comprise
nucleic acids encoding the peptides disclosed herein in an expressible
form. Herein, the phrase "in an expressible form" means that the
polynucleotide, when introduced into a cell, will be expressed in vivo as
a polypeptide that induces anti-tumor immunity. In an exemplified
embodiment, the nucleic acid sequence of the polynucleotide of interest
includes regulatory elements necessary for expression of the
polynucleotide. The polynucleotide(s) can be equipped so to achieve
stable insertion into the genome of the target cell (see, e.g., Thomas K
R & Capecchi M R, Cell 1987, 51: 503-12 for a description of homologous
recombination cassette vectors). See, e.g., Wolff et al., Science 1990,
247: 1465-8; U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118;
5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based delivery
technologies include "naked DNA", facilitated (bupivacaine, polymers,
peptide-mediated) delivery, cationic lipid complexes, and
particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g.,
U.S. Pat. No. 5,922,687).

[0112] The peptides of the invention can also be expressed by viral or
bacterial vectors. Examples of expression vectors include attenuated
viral hosts, such as vaccinia or fowlpox. This approach involves the use
of vaccinia virus, e.g., as a vector to express nucleotide sequences that
encode the peptide. Upon introduction into a host, the recombinant
vaccinia virus expresses the immunogenic peptide, and thereby elicits an
immune response. Vaccinia vectors and methods useful in immunization
protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector
is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et
al., Nature 1991, 351: 456-60. A wide variety of other vectors useful for
therapeutic administration or immunization e.g., adeno and
adeno-associated virus vectors, retroviral vectors, Salmonella typhi
vectors, detoxified anthrax toxin vectors, and the like, will be
apparent. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock
et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In Vivo 2000, 14:
571-85.

[0113] Delivery of a polynucleotide into a patient can be either direct,
in which case the patient is directly exposed to a
polynucleotide-carrying vector, or indirect, in which case, cells are
first transformed with the polynucleotide of interest in vitro, then the
cells are transplanted into the patient. Theses two approaches are known,
respectively, as in vivo and ex vivo gene therapies.

[0115] The method of administration can be oral, intradermal,
subcutaneous, intravenous injection, or such, and systemic administration
or local administration to the vicinity of the targeted sites finds use.
The administration can be performed by single administration or boosted
by multiple administrations. The dose of the polynucleotide in the
suitable carrier or cells transformed with the polynucleotide encoding
the peptides of this invention can be adjusted appropriately according to
the disease to be treated, age of the patient, weight, method of
administration, and such, and is ordinarily 0.001 mg to 1000 mg, for
example, 0.001 mg to 1000 mg, for example, 0.1 mg to 10 mg, and can be
administered once every a few days to once every few months. One skilled
in the art can appropriately select the suitable dose.

X. Methods Using the Peptides, Exosomes, APCs and CTLs

[0116] The peptides of the present invention and polynucleotides encoding
the peptides can be used for inducing APCs and CTLs. The exosomes and
APCs of the present invention can be also used for inducing CTLs. The
peptides, polynucleotides, exosomes and APCs can be used in combination
with any other compounds so long as the compounds do not inhibit their
CTL inducibility. Thus, any of the aforementioned pharmaceutical agents
of the present invention can be used for inducing CTLs, and in addition
thereto, those comprising the peptides and polynucleotides can be also be
used for inducing APCs as explained below.

[0117] (1) Method of Inducing Antigen-Presenting Cells (APCs)

[0118] The present invention provides methods of inducing APCs using the
peptides of this invention or polynulceotides encoding the peptides. The
induction of APCs can be performed as described above in section "VI.
Antigen-presenting cells". This invention also provides a method for
inducing APCs having a high level of CTL inducibility, the induction of
which has been also mentioned under the item of "VI. Antigen-presenting
cells", supra. Preferably, the present invention provides a method for
inducing APCs with high CTL inducibility, wherein the method comprises
the step of contacting the peptides of the invention with APCs or
introducing a polynucleotide encoding the peptides into APCs to produce
the APCs presenting the peptide of the present invention with HLA
antigens.

[0119] (2) Method of Inducing CTLs

[0120] Furthermore, the present invention provides methods for inducing
CTLs using the peptides of this invention, polynucleotides encoding the
peptides, or exosomes or APCs presenting the peptides. In a preferred
embodiment, the method comprises the step of contacting CD8+ T cells
with;

[0121] (a) APCs presenting the peptides of the present invention with HLA
antigens, or

[0122] (b) APCs which is induced by introducing a gene that comprises a
polynucleotide encoding the peptides of the present invention into an
antigen-presenting cell. When the peptides of this invention are
administered to a subject, CTL is induced in the body of the subject, and
the strength of the immune response targeting the tumor-associated
endothelia is enhanced. Alternatively, the peptides and polynucleotides
encoding the peptides can be used for an ex vivo therapeutic method, in
which subject-derived APCs, and CD8-positive cells, or peripheral blood
mononuclear leukocytes are contacted (stimulated) with the peptides of
this invention in vitro, and after inducing CTL, the activated CTL cells
are returned to the subject. For example, the method can comprise steps
of:

[0123] a: collecting APCs from subject:

[0124] b: contacting with the APCs of step a, with the peptide:

[0125] c: mixing the APCs of step b with CD8+ T cells, and
co-culturing for inducing CTLs: and

[0126] d: collecting C8+ T cells from the co-culture of step c.

[0127] Alternatively, according to the present invention, use of the
peptides of this invention for manufacturing a pharmaceutical composition
inducing CTLs is provided. Further, the present invention also provides
the peptide of the present invention for inducing CTLs.

[0128] The CD8+ T cells having cytotoxic activity obtained by step d
can be administered to the subject as a vaccine. The APCs to be mixed
with the CD8+ T cells in above step c can also be prepared by
transferring genes coding for the present peptides into the APCs as
detailed above in section "VI. Antigen-presenting cells"; but are not
limited thereto and any APC or exosome which effectively presents the
present peptides to the T cells can be used for the present method.

[0129] Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the present
invention, suitable methods and materials are described. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety. In case
of conflict, the present specification, including definitions, will
control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.

[0130] The following examples are presented to illustrate the present
invention and to assist one of ordinary skill in making and using the
same. The examples are not intended in any way to otherwise limit the
scope of the invention.

[0134] 9-mer and 10-mer peptides derived from TEM8 that bind to HLA-A*2402
and HLAA*0201 molecules were predicted using binding prediction software
"BIMAS" (http://www-bimas.cit.nih.gov/molbio/hla_bind), which algorithms
had been described by Parker K C et al. (J Immunol 1994, 152(1): 163-75)
and Kuzushima K et al. (Blood 2001, 98(6): 1872-81). These peptides were
synthesized by Sigma (Sapporo, Japan) according to a standard solid phase
synthesis method and purified by reversed phase high performance liquid
chromatography (HPLC). The purity (>90%) and the identity of the
peptides were determined by analytical HPLC and mass spectrometry
analysis, respectively. Peptides were dissolved in dimethylsulfoxide
(DMSO) at 20 mg/ml and stored at -80 degrees C.

[0135] In Vitro CTL Induction

[0136] Monocyte-derived dendritic cells (DCs) were used as
antigen-presenting cells (APCs) to induce cytotoxic T lymphocyte (CTL)
responses against peptides presented on human leukocyte antigen (HLA).
DCs were generated in vitro as described elsewhere (Nakahara S et al.,
Cancer Res 2003 Jul. 15, 63(14): 4112-8). Specifically, peripheral blood
mononuclear cells (PBMCs) isolated from a normal volunteer (HLA-A*2402 or
HLA-A*0201 positive) by Ficoll-Plaque (Pharmacia) solution were separated
by adherence to a plastic tissue culture dish (Becton Dickinson) so as to
enrich them as the monocyte fraction. The monocyte-enriched population
was cultured in the presence of 1000 U/ml of granulocyte-macrophage
colony-stimulating factor (GM-CSF) (R&D System) and 1000 U/ml of
interleukin (IL)-4 (R&D System) in AIM-V Medium (Invitrogen) containing
2% heat-inactivated autologous serum (AS). After 7 days of culture, the
cytokine-induced DCs were pulsed with 20 micrograms/ml of each of the
synthesized peptides in the presence of 3 micrograms/ml of
beta2-microglobulin for 3 hr at 37 degrees C. in AIM-V Medium. The
generated cells appeared to express DC-associated molecules, such as
CD80, CD83, CD86 and HLA class II, on their cell surfaces (data not
shown). These peptide-pulsed DCs were then inactivated by Mitomycin C
(MMC) (30 micrograms/ml for 30 min) and mixed at a 1:20 ratio with
autologous CD8+ T cells, obtained by positive selection with CD8 Positive
Isolation Kit (Dynal). These cultures were set up in 48-well plates
(Corning); each well contained 1.5×104 peptide-pulsed DCs,
3×105 CD8+ T cells and 10 ng/ml of IL-7 (R&D System) in 0.5 ml
of AIM-V/2% AS medium. Three days later, these cultures were supplemented
with IL-2 (CHIRON) to a final concentration of 20 IU/ml. On day 7 and 14,
the T cells were further stimulated with the autologous peptide-pulsed
DCs. The DCs were prepared each time by the same way described above. CTL
was tested against peptide-pulsed A24LCL cells after the 3rd round of
peptide stimulation on day 21 (Tanaka H et al., Br J Cancer 2001 Jan. 5,
84(1): 94-9; Umano Y et al., Br J Cancer 2001 Apr. 20, 84(8): 1052-7;
Uchida N et al., Clin Cancer Res 2004 Dec. 15, 10(24): 8577-86; Suda T et
al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci
2005 August, 96(8): 498-506).

[0144] For priming the peptide-specific CTLs, immunization was given using
100 mlcroliters vaccine mixture, which contains 50 microliters of HLA-A24
restricted peptide and 50 microliters of IFA per mouse. The vaccine was
injected s.c. into the right flank of mice for the first immunization on
day 0 and in the left flank for the second on the day 7. On day 14,
splenocytes from vaccinated mice were used as the responder cells, and
RLmale1 cells pulsed with or without peptides were used as the stimulator
cells for IFN-gamma ELISPOT assay.

[0145] In Vivo Antitumor Effects

[0146] Vaccination was done on days -7 and 0 using IFA-conjugated peptide.
On day O, CT26 cells (5×104 cells per mouse) were injected
s.c. into the right flank of BALB/c mice. Tumor growth was measured as
the product of two perpendicular diameters (mm 2).

[0147] Results

Prediction of HLA-A24 Binding Peptides Derived from TEM8

[0148] Table 1 shows the HLA-A*2402 and HLA-A*0201 binding peptides of
TEM8 in the order of high binding affinity. A total of 21 peptides with
potential HLA-A24 binding ability were selected and examined to determine
the epitope peptides (Table 1a), and a total of 53 peptides with
potential HLA-A2 binding ability were similarly selected and examined to
determine the epitope peptides (Table 1b, and Table 1c). Most of the
predicted peptides showed no-CTL induction. Therefore, in this invention
the peptide which showed CTL induction was focused upon.

[0149] CTL Induction with the Predicted Peptides from TEM8 Restricted with
HLA-A*2402 or HLA-A*0201 and Establishment for CTL Lines Stimulated with
TEM8 Derived Peptides

[0150] CTLs for those peptides derived from TEM8 were generated according
to the protocols as described in "Materials and Methods". Peptide
specific CTL activity was determined by IFN-gamma ELISPOT assay (FIG.
1a-i). It showed that TEM8-A24-9-39 (SEQ ID NO: 3), TEM8-A24-9-277 (SEQ
ID NO: 4), TEM8-A24-10-277 (SEQ ID NO: 9), TEM8-A02-9-337 (SEQ ID NO:
23), TEM8-A02-9-338 (SEQ ID NO: 25), TEM8-A02-9-278 (SEQ ID NO: 30),
TEM8-A02-10-338 (SEQ ID NO: 60), TEM8-A02-10-265 (SEQ ID NO: 63) and
TEM8-A02-10-333 (SEQ ID NO: 68) demonstrated potent IFN-gamma production
as compared to the control wells. Furthermore, the cells in the positive
well number #5 stimulated with SEQ ID NO: 3, #6 with SEQ ID NO: 4, #3
with SEQ ID NO: 9, #3 with SEQ ID NO: 23, #6 with SEQ ID NO: 25, #3 with
SEQ ID NO: 30, #2 with SEQ ID NO: 60, #5 with SEQ ID NO: 63 and #4 with
SEQ ID NO: 68 were expanded and established CTL lines. CTL activity of
those CTL lines was determined by IFN-gamma ELISA assay (FIG. 2a-i). It
showed that all CTL lines demonstrated potent IFN-gamma production
against the target cells pulsed with corresponding peptide as compared to
target cells without peptide pulse. On the other hand, no CTL lines could
be established by stimulation with other peptides shown in Table 1,
despite those peptide had possible binding activity with HLA-A*2402 or
HLA-A*0201. For example, typical negative data of CTL response stimulated
with TEM8-A02-9-207 (SEQ ID NO: 46) was shown in FIG. 1j and FIG. 2j. As
a result, it indicated that nine peptides derived from TEM8 were screened
as the peptides could induce potent CTL lines.

[0151] Establishment of CTL Clones Against TEM8 Specific Peptides

[0152] Furthermore, CTL clones were established by limiting dilution from
each CTL lines as described in under the item of "Materials and Methods",
and IFN-gamma production from CTL clones against target cells pulsed
peptide were determined by IFN-gamma ELISA assay. Potent IFN-gamma
production were determined from CTL clones stimulated with SEQ ID NO: 4,
SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 63 in FIG. 3.

[0154] The established CTL lines or clones raised against these peptides
were examined for their ability to recognize target cells that
endogenously express TEM8 and HLAA*2402 molecule. Specific CTL activity
against COS7 cells which were transfected with both the full length of
TEM8 and HLA-A*2402 molecule genes (a specific model for the target cells
that endogenously express TEM8 and HLA-A*2402 genes) was tested using the
CTL lines or clones raised by corresponding peptide as the effector
cells. COS7 cells transfected with either the full length of TEM8 genes
or HLA-A* 2402, were prepared as control. The CTLs showed potent CTL
activity against COS7 cells transfected with both TEM8 and HLA-A24 in
FIG. 4a. On the other hand, no significant specific CTL activity was
detected against the controls. Furthermore, established CTL lines or
clones raised against HLA-A2 restricted peptides were also examined for
their ability to recognize target cells that endogenously express TEM8
and HLA-A*0201 molecule. Specific CTL activity against COS7 cells which
were transfected with both the full length of TEM8 and HLA-A*0201
molecule genes was tested. The CTL lines or clones induced by
corresponding HLA-A2 restricted peptide were used as the effector cells.
COS7 cells transfected with either full length of TEM8 gene or HLA-A*0201
gene were used as control. The CTLs stimulated with SEQ ID NO: 63 showed
potent CTL activity against COS7 cells transfected with both TEM8 and
HLA-A2 in FIG. 4b. On the other hand, no significant specific CTL
activity was detected against the controls. Thus, these data clearly
demonstrated that SEQ ID NO: 4 and SEQ ID NO: 63 were naturally expressed
on the target cells with HLA-A*2402 or HLA-A*0201 molecule and were
recognized by the CTLs. Furthermore, it indicated that those two peptides
derived from TEM8 are epitope peptides could induce CTLs and it may be
available to apply the cancer vaccines for patients with TEM8 expressing
cells.

[0155] Immunogenicity of Epitope Peptide in BALB/c Mice

[0156] Immunization of SEQ ID NO: 4 to BALB/c mice was performed to
evaluate the immunogenicity of TEM8 epitope peptides After second
injection of the peptide conjugated with IFA, peptide specific CTL
activity was determined by IFN-gamma ELISPOT assay. When the splenocytes
harvested from the vaccinated mice were used as responder cells, potent
IFN-gamma production was specifically detected In FIG. 5a, IFN-gamma
production specific to SEQ ID NO: 4 were detected from four of five mice
(M1, M3, M4 and M5) but not in control mice (N1 and N2). These data
indicated that SEQ ID NO: 4 induced specific CTLs against peptide-pulsed
target cells in vivo.

[0157] Antitumor Effects of Vaccination of TEM8 Epitope Peptide

[0158] To examine the antitumor effects using the peptide, in vivo
anti-tumor model was examined using CT26 tumor cell lines. Administration
of SEQ ID NO: 4 was performed on the day -7 and 0, and CT26 colorectal
cancer cells were injected s.c. into BALB/c mice on day 0. Tumor growth
apparently reduced in the mice vaccinated of SEQ ID NO: 4 compared with
the control mice (FIG. 5b). It showed the statistically significant
difference with suppression of tumor growth in the mice with vaccination
using SEQ ID NO: 4 (P<0.05).

[0159] Homology Analysis of Antigen Peptides

[0160] It was demonstrated that the CTLs stimulated with the following
peptides respectively, showed significant and specific CTL activity.

[0161] TEM8-A24-9-39 (SEQ ID NO: 3),

[0162] TEM8-A24-9-277 (SEQ ID NO: 4),

[0163] TEM8-A24-10-277 (SEQ ID NO: 9),

[0164] TEM8-A02-9-337 (SEQ ID NO: 23),

[0165] TEM8-A02-9-338 (SEQ ID NO: 25),

[0166] TEM8-A02-9-278 (SEQ ID NO: 30),

[0167] TEM8-A02-10-338 (SEQ ID NO: 60),

[0168] TEM8-A02-10-265 (SEQ ID NO: 63) and

[0169] TEM8-A02-10-333 (SEQ ID NO: 68),

[0170] This result may be due to the fact that the sequences of the
peptides are homologous to peptides derived from other molecules that are
known to sensitize the human immune system. To exclude this possibility,
homology analysis was performed for these peptide sequences using as
queries the BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi)
which revealed no sequence with significant homology. The results of
homology analyses indicate that the sequences of TEM8-A24-9-39 (SEQ ID
NO: 3), TEM8-A24-9-277 (SEQ ID NO: 4), TEM8-A24-10-277 (SEQ ID NO: 9),
TEM8-A02-9-337 (SEQ ID NO: 23), TEM8-A02-9-338 (SEQ ID NO: 25),
TEM8-A02-9-278 (SEQ ID NO: 30), TEM8-A02-10-338 (SEQ ID NO: 60),
TEM8-A02-10-265 (SEQ ID NO: 63) and TEM8-A02-10-333 (SEQ ID NO: 68),
respectively, are unique and thus, there is little possibility, to our
best knowledge, that these molecules raise an unintended immunologic
response to some unrelated molecule.

[0171] Finally, novel HLA-A*2402 or A*0201 epitope peptides derived from
TEM8 were identified. Furthermore, it was demonstrated that epitope
peptides of TEM8 are applicable for cancer immunotherapy.

INDUSTRIAL APPLICABILITY

[0172] The present invention provides novel peptides, which induce CTLs
targeting endothelial cells formed in a wide range of diseases associated
with angiogenesis, and which peptides are extremely effective as
vaccines. The present invention also provides pharmaceuticals for
treating and prevention of diseases associated with angiogenesis, such as
tumors, which comprise any of these peptides as the active ingredient.
According to the present invention, the size of the peptide required for
inducing immunity is very small (e.g., 9 to 10 amino acid residues).
Therefore, the present invention is particularly advantageous in that the
synthesis and purification of the peptides can be quite easily performed.

[0173] All publications, patents, and patent applications cited herein are
incorporated into the present description by reference.